Citalopram and escitalopram plasma drug and metabolite concentrations: genome-wide associations.

AIMS: Citalopram (CT) and escitalopram (S-CT) are among the most widely prescribed selective serotonin reuptake inhibitors used to treat major depressive disorder (MDD). We applied a genome-wide association study to identify genetic factors that contribute to variation in plasma concentrations of CT or S-CT and their metabolites in MDD patients treated with CT or S-CT. METHODS: Our genome-wide association study was performed using samples from 435 MDD patients. Linear mixed models were used to account for within-subject correlations of longitudinal measures of plasma drug/metabolite concentrations (4 and 8 weeks after the initiation of drug therapy), and single-nucleotide polymorphisms (SNPs) were modelled as additive allelic effects. RESULTS: Genome-wide significant associations were observed for S-CT concentration with SNPs in or near the CYP2C19 gene on chromosome 10 (rs1074145, P = 4.1 × 10(-9) ) and with S-didesmethylcitalopram concentration for SNPs near the CYP2D6 locus on chromosome 22 (rs1065852, P = 2.0 × 10(-16) ), supporting the important role of these cytochrome P450 (CYP) enzymes in biotransformation of citalopram. After adjustment for the effect of CYP2C19 functional alleles, the analyses also identified novel loci that will require future replication and functional validation. CONCLUSIONS: In vitro and in vivo studies have suggested that the biotransformation of CT to monodesmethylcitalopram and didesmethylcitalopram is mediated by CYP isozymes. The results of our genome-wide association study performed in MDD patients treated with CT or S-CT have confirmed those observations but also identified novel genomic loci that might play a role in variation in plasma levels of CT or its metabolites during the treatment of MDD patients with these selective serotonin reuptake inhibitors.

Effects of the CYP2B6*6 allele on catalytic properties and inhibition of CYP2B6 in vitro: implication for the mechanism of reduced efavirenz metabolism and other CYP2B6 substrates in vivo.

The mechanism by which CYP2B6*6 allele alters drug metabolism in vitro and in vivo is not fully understood. To test the hypothesis that altered substrate binding and/or catalytic properties contribute to its functional consequences, efavirenz 8-hydroxylation and bupropion 4-hydroxylation were determined in CYP2B6.1 and CYP2B6.6 proteins expressed without and with cytochrome b5 (Cyt b5) and in human liver microsomes (HLMs) obtained from liver tissues genotyped for the CYP2B6*6 allele. The susceptibility of the variant protein to inhibition was also tested in HLMs. Significantly higher V(max) and K(m) values for 8-hydroxyefavirenz formation and ∼2-fold lower intrinsic clearance (Cl(int)) were noted in expressed CYP2B6.6 protein (-b5) compared with that of CYP2B6.1 protein (-b5); this effect was abolished by Cyt b5. The V(max) and Cl(int) values for 4-hydroxybupropion formation were significantly higher in CYP2B6.6 than in CYP2B6.1 protein, with no difference in K(m), whereas coexpression with Cyt b5 reversed the genetic effect on these kinetic parameters. In HLMs, CYP2B6*6/*6 genotype was associated with markedly lower V(max) (and moderate increase in K(m)) and thus lower Cl(int) values for efavirenz and bupropion metabolism, but no difference in catalytic properties was noted between CYP2B6*1/*1 and CYP2B6*1/*6 genotypes. Inhibition of efavirenz 8-hydroxylation by voriconazole was significantly greater in HLMs with the CYP2B6*6 allele (K(i) = 1.6 ± 0.8 µM) than HLMs with CYP2B6*1/*1 genotype (K(i) = 3.0 ± 1.1 µM). In conclusion, our data suggest the CYP2B6*6 allele influences metabolic activity by altering substrate binding and catalytic activity in a substrate- and Cyt b5-dependent manner. It may also confer susceptibility to inhibition.

Stereoselective and regiospecific hydroxylation of ketamine and norketamine.

The objective was to determine the cytochrome P450s (CYPs) responsible for the stereoselective and regiospecific hydroxylation of ketamine [(R,S)-Ket] to diastereomeric hydroxyketamines, (2S,6S;2R,6R)-HK (5a) and (2S,6R;2R,6S)-HK (5b) and norketamine [(R,S)-norKet] to hydroxynorketamines, (2S,6S;2R,6R)-HNK (4a), (2S,6R;2R,6S)-HNK (4b), (2S,5S;2R,5R)-HNK (4c), (2S,4S;2R,4R)-HNK (4d), (2S,4R;2R,4S)-HNK (4e), (2S,5R;2R,5S)-HNK (4f). The enantiomers of Ket and norKet were incubated with characterized human liver microsomes (HLMs) and expressed CYPs. Metabolites were identified and quantified using LC/MS/MS and apparent kinetic constants estimated using single-site Michaelis-Menten, Hill or substrate inhibition equation. 5a was predominantly formed from (S)-Ket by CYP2A6 and N-demethylated to 4a by CYP2B6. 5b was formed from (R)- and (S)-Ket by CYP3A4/3A5 and N-demethylated to 4b by multiple enzymes. norKet incubation produced 4a, 4c and 4f and minor amounts of 4d and 4e. CYP2A6 and CYP2B6 were the major enzymes responsible for the formation of 4a, 4d and 4f, and CYP3A4/3A5 for the formation of 4e. The 4b metabolite was not detected in the norKet incubates. 5a and 4b were detected in plasma samples from patients receiving (R,S)-Ket, indicating that 5a and 5b are significant Ket metabolites. Large variations in HNK concentrations were observed suggesting that pharmacogenetics and/or metabolic drug interactions may play a role in therapeutic response.

Contribution of N-glucuronidation to efavirenz elimination in vivo in the basal and rifampin-induced metabolism of efavirenz.

In this study, the contribution of efavirenz N-glucuronidation to efavirenz elimination in vivo was assessed. In a two-period placebo-controlled crossover trial design, a single 600-mg oral dose of efavirenz was administered to healthy volunteers (n = 10) pretreated with placebo pills or 600 mg/day rifampin orally for 10 days. Urine and plasma concentrations of efavirenz and 8-hydroxyefavirenz were measured by the liquid chromatography-tandem mass spectrometry method after enzymatic hydrolysis with ß-glucuronidase (conjugated and unconjugated) and without enzymatic hydrolysis (unconjugated). Pharmacokinetic parameters of efavirenz within the placebo- or rifampin-treated group obtained after enzymatic hydrolysis did not show any statistically significant difference compared with those obtained without enzymatic hydrolysis (P > 0.05; paired t test, two-tailed). The amount of efavirenz excreted over 12 h was significantly larger after enzymatic hydrolysis in both the placebo (P = 0.007) and rifampin (P = 0.0001) treatment groups, supporting the occurrence of direct N-glucuronidation of efavirenz, but the relevance of this finding is limited because the amount of efavirenz excreted as unchanged or conjugated in urine is less than 1% of the dose administered. In both the placebo- and rifampin-treated groups, plasma concentrations of 8-hydroxyefavirenz and the amount excreted over 12 h were significantly larger (P < 0.00001) after enzymatic hydrolysis than without enzymatic hydrolysis. These findings suggest that although the occurrence of direct efavirenz N-glucuronidation is supported by the urine data, the abundance of efavirenz N-glucuronide in plasma is negligible and that the contribution of the N-glucuronidation pathway to the overall clearance of efavirenz seems minimal.

Microfabricated electrochemical detector for high-performance liquid chromatography.

A microfabricated electrochemical cell has been developed as a disposable detector for flow injection analysis (FIA) and high-performance liquid chromatography (HPLC). The simplicity of the fabrication process allows this detector to be used as a low-cost, disposable device that can be replaced easily if its performance degrades rather than disassembling the detector and polishing the electrode surface, which is the usual procedure. The detector consists of thin film-metal electrodes-platinum working electrode, platinum auxiliary electrode, and silver/silver chloride coated on Pt reference electrode-deposited on a polyimide substrate with a locking layer of chromium in between. A microfluidic cover made of polyimide directs the solution flow across the electrodes. The detector was evaluated with FIA of ferrocyanide and HPLC of ascorbic acid and acetaminophen and a mixture of two pharmaceutical compounds-dextrorphan and levallorphan-with acetaminophen internal standard. The HPLC calibration curves showed good linearity (R(2) > 0.99). Limits of detection were 1 nM for acetaminophen, 1 nM for ascorbic acid, 50 nM for dextrorphan, and 80 nM for levallorphan. When detected with a commercial detector dextrorphan and levallorphan had lower limits of detection, 3 and 5 nM, respectively. Chromatograms of the mixture were comparable to those obtained with a commercial detector. The detector could be used continuously for about 48 h with FIA and about 10-20 h with HPLC after which performance gradually degraded as the AgCl on the reference electrode dissolved causing loss of potential control.

Efavirenz primary and secondary metabolism in vitro and in vivo: identification of novel metabolic pathways and cytochrome P450 2A6 as the principal catalyst of efavirenz 7-hydroxylation.

Efavirenz primary and secondary metabolism was investigated in vitro and in vivo. In human liver microsome (HLM) samples, 7- and 8-hydroxyefavirenz accounted for 22.5 and 77.5% of the overall efavirenz metabolism, respectively. Kinetic, inhibition, and correlation analyses in HLM samples and experiments in expressed cytochrome P450 show that CYP2A6 is the principal catalyst of efavirenz 7-hydroxylation. Although CYP2B6 was the main enzyme catalyzing efavirenz 8-hydroxylation, CYP2A6 also seems to contribute. Both 7- and 8-hydroxyefavirenz were further oxidized to novel dihydroxylated metabolite(s) primarily by CYP2B6. These dihydroxylated metabolite(s) were not the same as 8,14-dihydroxyefavirenz, a metabolite that has been suggested to be directly formed via 14-hydroxylation of 8-hydroxyefavirenz, because 8,14-dihydroxyefavirenz was not detected in vitro when efavirenz, 7-, or 8-hydroxyefavirenz were used as substrates. Efavirenz and its primary and secondary metabolites that were identified in vitro were quantified in plasma samples obtained from subjects taking a single 600-mg oral dose of efavirenz. 8,14-Dihydroxyefavirenz was detected and quantified in these plasma samples, suggesting that the glucuronide or the sulfate of 8-hydroxyefavirenz might undergo 14-hydroxylation in vivo. In conclusion, efavirenz metabolism is complex, involving unique and novel secondary metabolism. Although efavirenz 8-hydroxylation by CYP2B6 remains the major clearance mechanism of efavirenz, CYP2A6-mediated 7-hydroxylation (and to some extent 8-hydroxylation) may also contribute. Efavirenz may be a valuable dual phenotyping tool to study CYP2B6 and CYP2A6, and this should be further tested in vivo.

In vitro and in vivo oxidative metabolism and glucuronidation of anastrozole.

AIMS: Little information is available regarding the metabolic routes of anastrozole and the specific enzymes involved. We characterized anastrozole oxidative and conjugation metabolism in vitro and in vivo. METHODS: A sensitive LC-MS/MS method was developed to measure anastrozole and its metabolites in vitro and in vivo. Anastrozole metabolism was characterized using human liver microsomes (HLMs), expressed cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs). RESULTS: Hydroxyanastrozole and anastrozole glucuronide were identified as the main oxidative and conjugated metabolites of anastrozole in vitro, respectively. Formation of hydroxyanastrozole from anastrozole was markedly inhibited by CYP3A selective chemical inhibitors (by >90%) and significantly correlated with CYP3A activity in a panel of HLMs (r= 0.96, P= 0.0005) and mainly catalyzed by expressed CYP3A4 and CYP3A5. The K(m) values obtained from HLMs were also close to those from CYP3A4 and CYP3A5. Formation of anastrozole glucuronide in a bank of HLMs was correlated strongly with imipramine N-glucuronide, a marker of UGT1A4 (r= 0.72, P < 0.0001), while expressed UGT1A4 catalyzed its formation at the highest rate. Hydroxyanastrozole (mainly as a glucuronide) and anastrozole were quantified in plasma of breast cancer patients taking anastrozole (1 mg day⁻¹); anastrozole glucuronide was less apparent. CONCLUSION: Anastrozole is oxidized to hydroxyanastrozole mainly by CYP3A4 (and to some extent by CYP3A5 and CYP2C8). Once formed, this metabolite undergoes glucuronidation. Variable activity of CYP3A4 (and probably UGT1A4), possibly due to genetic polymorphisms and drug interactions, may alter anastrozole disposition and its effects in vivo.

CYP2D6 genotypes, endoxifen levels, and disease recurrence in 224 Filipino and Vietnamese women receiving adjuvant tamoxifen for operable breast cancer.

BACKGROUND: While tamoxifen activity is mainly due to endoxifen and the concentration of this active metabolite is, in part, controlled by CYP2D6 metabolic status, clinical correlative studies have produced mixed results. FINDINGS: In an exploratory study, we determined the CYP2D6 metabolic status and plasma concentrations of endoxifen among 224 Filipino and Vietnamese women participating in a clinical trial of adjuvant hormonal therapy for operable breast cancer. We further conducted a nested-case-control study among 48 women (half with recurrent disease, half without) investigating the relationship of endoxifen concentrations and recurrence of disease. We found a significant association of reduced endoxifen plasma concentrations with functionally important CYP2D6 genotypes. High endoxifen concentrations were associated with higher risk of recurrence; with a quadratic trend fitted to a stratified Cox proportional hazards regression model, the likelihood ratio p-value was 0.002. The trend also showed that in 8 out of 9 pairs with low endoxifen concentrations, the recurrent case had lower endoxifen levels than the matched control. CONCLUSIONS: This exploratory analysis suggests that there is an optimal range for endoxifen concentrations to achieve favorable effects as adjuvant therapy. In particular, at higher concentrations (>70 ng.ml), endoxifen may promote recurrence.

Genotype-guided tamoxifen dosing increases active metabolite exposure in women with reduced CYP2D6 metabolism: a multicenter study.

PURPOSE: We examined the feasibility of using CYP2D6 genotyping to determine optimal tamoxifen dose and investigated whether the key active tamoxifen metabolite, endoxifen, could be increased by genotype-guided tamoxifen dosing in patients with intermediate CYP2D6 metabolism. PATIENTS AND METHODS: One hundred nineteen patients on tamoxifen 20 mg daily ≥ 4 months and not on any strong CYP2D6 inhibiting medications were assayed for CYP2D6 genotype and plasma tamoxifen metabolite concentrations. Patients found to be CYP2D6 extensive metabolizers (EM) remained on 20 mg and those found to be intermediate (IM) or poor (PM) metabolizers were increased to 40 mg daily. Eighty-nine evaluable patients had tamoxifen metabolite measurements repeated 4 months later. RESULTS: As expected, the median baseline endoxifen concentration was higher in EM (34.3 ng/mL) compared with either IM (18.5 ng/mL; P = .0045) or PM (4.2 ng/mL; P < .001). When the dose was increased from 20 mg to 40 mg in IM and PM patients, the endoxifen concentration rose significantly; in IM there was a median intrapatient change from baseline of +7.6 ng/mL (-0.6 to 23.9; P < .001), and in PM there was a change of +6.1 ng/mL (2.6 to 12.5; P = .020). After the dose increase, there was no longer a significant difference in endoxifen concentrations between EM and IM patients (P = .84); however, the PM endoxifen concentration was still significantly lower. CONCLUSION: This study demonstrates the feasibility of genotype-driven tamoxifen dosing and demonstrates that doubling the tamoxifen dose can increase endoxifen concentrations in IM and PM patients.